Brucella species are Gram-negative facultative intracellular bacteria that induce chronic infectious disease and are potential pathogens for bioterrorism. However, little is known regarding Brucella genes responsible for intracellular survival and pathogenesis. B. abortus, B. melitensis, and B. suis are pathogenic to humans, while B. ovis and B. neotomae are nonpathogenic to humans, and B. canis human infections are rare. Limited genome diversity exists among Brucella species (1); however, considerable differences in pathogenicity occur. Therefore, identifying genome differences among Brucella species would reveal genes or gene clusters that may contribute to intracellular survival and pathogenesis. Recently, we have used a microarray of the complete B. melitensis genome, the species highly pathogenic to humans, to hybridize DNA from other Brucella species, and the results revealed clusters (genomic islands, GIs) in the B. melitensis 16M genome that were missing in the nonpathogenic species. The genomic context of these newly identified islands indicates that they are horizontally acquired and share resemblance to several pathogenicity islands from other bacteria. A GI-5 deletion mutant is attenuated in IRF-1(-/-) mice, and many of the genes within GI-5 are associated with virulence in other bacteria. We hypothesize that genetic information within GI-5 contributes to establishing an intracellular niche and persistence of Brucella. Intracellular Brucella are subjected to host antimicrobial peptides, and GI-5 contains many oligopeptide transport systems that may respond to these host antimicrobial peptides. Our preliminary studies position us to submit this R21 application to gather additional data identifying regions, genes and operons within GI-5 that contribute to bacterial pathogenicity prior to an R01 application. We have the following specific aims: Aim 1. We will construct mutants within genomic island-5. a) We will delete GI-5 in bioluminescent B. melitensis to determine its overall importance to virulence; b) We will perform selective deletions and complementation of genes/operons within GI-5. Aim 2. We will determine the functional importance of GI-5 in macrophages and mice, a) We will determine if GI-5 and genes/operons within GI-5 have functional importance for Brucella survival in macrophages. b) We will compare the in vivo dissemination and persistence of GI-5 mutants to pathogenic B. melitensis in mice using biophotonic imaging, c) We will determine the susceptibility of the GI-5 mutant to macrophage antimicrobial mechanisms. Aim 3. We will determine B. melitensis transcription in macrophages that may lead to intracellular survival strategies, a) We will determine the bacterial transcript levels of pathogenic B. melitensis in macrophages. [unreadable] [unreadable] [unreadable]